A very important task in the ongoing search for new clinically useful drugs is the generation of large numbers of structurally diverse compounds. These molecules are required for screening by high-throughput bioassays in the discovery of new lead drug candidates. Combinatorial biosynthesis, in which nature's chemical capabilities are exploited in a combinatorial "mix-and-match" fashion, has generated libraries of novel molecules representing great structural diversity which are not available naturally or generated through combinatorial synthesis. The marine bacterium "Streptomyces maritimus" is uniquely capable of naturally producing a diverse series of structurally uncommon bactereostatic polyketides known as the enterocins and wailupemycins. Cloning, sequencing, and heterologous expression of the single biosynthesis gene cluster (enc) for these molecules revealed an unprecedented iterative type II polyketide synthase (PKS) system. Polyketide structural variability is probably achieved by the lack of a dedicated cyclase and the action of a rare enzyme-mediated Favorskii rearrangement. The novel architecture of this natural PKS gene set furnishes insight into engineering molecular diversity through genetic recombination and provides the major rationale for the proposed project. In this grant application, we propose to continue our biosynthetic analysis of the enterocin family at the chemical, biochemical and genetic levels. Our primary goal is to fully characterize this natural biosynthetically diverse pathway in order to engineer mutant organisms harboring and expressing altered gene clusters in which specific biosynthetic genes have been deleted, added or replaced with homologous genes from other biosynthetic pathways. The resulting recombinant will be chemically analyzed for the production of polyketide analogs and assayed for biological activities. Biosynthetic enzymes that will be exploited in recombinant systems include: EncM, a flavin-dependent oxygenase that is putatively involved in the derailment of the minimal enc PKS from generating aromatic endproducts, the enzymes involved in the synthesis and incorporation of the rare benzoyl-CoA PKS starter unit, the regiospecific P450 monooxygenase EncR, and the substrate tolerant methyltransferase EncK. Secondly, the "S. Maritimus" enc genes and their sequences will be used to analyze an enterocin-containing marine invertebrate and its associated microflora for the presence of homologous genes in order to model marine microbial-invertebrate symbiosis.